Metabolic studies of cells expressing the mutant protein demonstrate that this mutation causes an alteration in processing resulting in a change in the ratio of glycosylated forms observed and an overall decrease in the stability of the prion protein. Further, the alterations we observed are evident in samples of brain from affected individuals. However, there are several limitations to the cell culture system. First, the metabolism of the prion protein seems to depend on the cell line in which is expressed; human neuroblastoma cells yield different results than Chinese hamster ovary cells. Thus, it is important to try to evaluate its metabolism in a "normal" cell. Second, some of the mutations affect very specific cell types, for example the thalamic neurons in fatal familial insomnia (Ffl). To address questions of selective vulnerability it is necessary to produce the intact organ with all the specialized cells. Third, to assess the role, if any, of other cell types also requires production of a whole animal model. Finally, and perhaps of the greatest significance is that the inherited prion diseases are expressed in an age dependent manner which has yet to be recapitulated in a monotypic cell culture system. For these reasons, we propose to develop a transgenic mouse model of both the codon 129 methionine variant (Ffl) and the 129 valine variant 178CJD. The transgenic animals will be evaluated for the metabolic defect observed in the human brain first by analyzing the glycoforms of prion protein produced in different brain regions and then by analyzing metabolism of the mutant protein in primary cultures of isolated cell types (e.g. neurons, astrocytes, etc.) Or in mixed cultures. These studies will be followed by experiments of infection of the transgenic animals with scrapie and by the analysis of the production of pathogenic PrP and the disease course of animals expressing normal or mutant PrP to determine the relative role of the inoculum and the endogenous genotype in determining the disease. The data generated in this Project will aid in our understanding of inherited as well as sporadic neurodegenerative conditions since it is likely that propagation of disease through the brain employs similar mechanisms once the disease process has been initiated. In particular, development of this animal model system will provide insight into the age related component(s) of neurodegenerative disease, something that can not be approached in cell culture models.